*Protein ID (NP or XP #) or Wolbachia#: *Organism (including strain): M. tuberculosis
*/Disease Information (sort of like the Intro to your Mini Research Write up): Tuberculosis (TB) is a contagious- infectious disease that is either active or dormant depending on the individual’s ability to defend itself against the pathogen. This causes those in developing countries, young/aging populations and immunocompromised to be most at risk [1]. Active TB takes two additional forms- pulmonary and extrapulmonary (outside the lungs), with extrapulmonary being noninfectious. An individual infected with active pulmonary tuberculosis is able to spread the pathogen through the air by sneezing, coughing or talking. Repeated contact may be necessary for infection, but only about 5% of infected individuals actually develop TB [2]. Despite this roughly a third of the world’s population is estimated to be infected with Mycobacterium tuberculosis, the pathogen responsible for TB, leading to approximately 9 million new cases and 3 million deaths per year [1].
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: Essential Reference: Comparative protein modeling of methionine S-adenosyltransferase (MAT) enzyme from Mycobacterium tuberculosis: a potential target for antituberculosis drug discovery
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.5 *EC#: 2.5.1.6 (methionine adenosyltransferase) same-same
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836674/#R7 -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): Yes, has crystal structure
Purification Method: Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Methionine added to beginning of sequence for E coli
MVSEKGRLFTSESVTEGHPDKICDAISDSVLDALLAADPRSRVAVETLVTTGQVHVVGEVT
TSAKEAFADITNTVRARILEIGYDSSDKGFDGATCGVNIGIGAQSPDIAQGVDTAHEARV
EGAADPLDSQGAGDQGLMFGYAINATPELMPLPIALAHRLSRRLTEVRKNGVLPYLRPDG
KTQVTIAYEDNVPVRLDTVVISTQHAADIDLEKTLDPDIREKVLNTVLDDLAHETLDAST
VRVLVNPTGKFVLGGPMGDAGLTGRKIIVDTYGGWARHGGGAFSGKDPSKVDRSAAYAMR
WVAKNVVAAGLAERVEVQVAYAIGKAAPVGLFVETFGTETEDPVKIEKAIGEVFDLRPGA
IIRDLNLLRPIYAPTAAYGHFGRTDVELPWEQLDKVDDLKRAI Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer: 5’ TACTTCCAATCCATGGTTTCTGAAAAAGGTCG 3’ 32 bp GC Content 40.6% 0 mM Mg2+ Tm 60.3oC1.5 mM Mg2+ Tm 68.1oC 2 mM Mg2+ Tm 68.5oC 4 mM Mg2+ Tm 69.4oC 6 mM Mg2+ Tm 69.9oC
Reverse Primer:
5’ TATCCACCTTTACTGTTAGATCGCACGTTTGAGAT 3’ 32 bp GC Content 40% 0 mM Mg2+ Tm 60.7oC1.5 mM Mg2+ Tm 68.6oC 2 mM Mg2+ Tm 69oC 4 mM Mg2+ Tm 69.8oC 6 mM Mg2+ Tm 70.3oC
References:
[1] Ducati, R. G.; Ruffino-Netto, A.; Basso, L. A.; Santos, D. S., The resumption of consumption -- a review on tuberculosis. Mem Inst Oswaldo Cruz2006,101 (7), 697-714.
S-adenosylmethionine synthetase
*NCBI Gene # or RefSeq#:
Tuberculist / Rv1392
*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain):
M. tuberculosis
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis (TB) is a contagious- infectious disease that is either active or dormant depending on the individual’s ability to defend itself against the pathogen. This causes those in developing countries, young/aging populations and immunocompromised to be most at risk [1]. Active TB takes two additional forms- pulmonary and extrapulmonary (outside the lungs), with extrapulmonary being noninfectious. An individual infected with active pulmonary tuberculosis is able to spread the pathogen through the air by sneezing, coughing or talking. Repeated contact may be necessary for infection, but only about 5% of infected individuals actually develop TB [2]. Despite this roughly a third of the world’s population is estimated to be infected with Mycobacterium tuberculosis, the pathogen responsible for TB, leading to approximately 9 million new cases and 3 million deaths per year [1].
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=6457
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: Essential Reference:
Comparative protein modeling of methionine S-adenosyltransferase (MAT) enzyme from Mycobacterium tuberculosis: a potential target for antituberculosis drug discovery
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
0.5
*EC#:
2.5.1.6 (methionine adenosyltransferase) same-same
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836674/#R7
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): Yes, has crystal structure
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids:
261 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
55.2%
CDS Gene:
ATGGTTTCTGAAAAAGGTCGTCTGTTTACGTCTGAGTCTGTTACGGAAGGCCATCCTGACAAAATTTGCGACG
CTATCTCTGACTCTGTACTGGATGCACTCCTGGCGGCAGACCCTCGTTCTCGTGTTGCGGTTGAGACTCTCGT
AACCACCGGTCAGGTTCACGTTGTTGGTGAAGTTACCACCTCTGCAAAAGAAGCGTTTGCGGATATCACTAAC
ACGGTCCGTGCGCGTATTCTGGAAATTGGCTACGACTCTTCTGACAAGGGTTTCGATGGTGCGACGTGCGGC
GTGAACATCGGTATCGGTGCGCAGTCTCCTGATATTGCGCAGGGTGTTGACACCGCACACGAAGCGCGTGTG
GAAGGTGCTGCAGATCCTCTGGACTCTCAGGGTGCCGGTGACCAGGGCCTGATGTTCGGTTACGCAATCAAT
GCGACCCCTGAACTGATGCCGCTCCCGATCGCGCTGGCCCATCGCCTGTCTCGTCGTCTCACTGAGGTTC
GTAAAAACGGTGTTCTCCCGTACCTCCGCCCAGATGGTAAGACCCAGGTTACTATCGCGTACGAGGATAACGT
TCCGGTTCGTCTCGACACTGTGGTAATCTCTACCCAGCACGCGGCTGACATCGACCTGGAGAAAACCCTCGA
TCCGGACATCCGCGAGAAAGTTCTCAACACTGTCCTGGATGACCTCGCACATGAGACGCTGGACGCATCCAC
CGTTCGTGTTCTGGTAAATCCTACGGGCAAATTCGTTCTGGGTGGTCCGATGGGTGATGCGGGTCTGACTGG
TCGCAAGATCATCGTAGACACCTACGGCGGTTGGGCCCGTCACGGTGGTGGTGCGTTCAGCGGCAAAGACC
CGTCTAAGGTTGACCGTTCTGCGGCCTATGCCATGCGTTGGGTTGCGAAAAACGTCGTGGCAGCAGGTCTGG
CGGAGCGTGTAGAAGTTCAAGTCGCGTATGCAATCGGTAAAGCGGCACCTGTTGGTCTGTTCGTTGAAACTTT
CGGCACCGAAACGGAAGATCCGGTAAAAATCGAAAAAGCGATTGGTGAGGTTTTTGATCTGCGTCCGGGTGC
CATCATTCGTGATCTGAATCTGCTCCGTCCAATCTACGCGCCGACCGCAGCGTACGGCCACTTTGGTCGTACC
GACGTTGAACTGCCGTGGGAACAACTGGACAAGGTCGACGATCTCAAACGTGCGATCTAA
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGGTTTCTGAAAAAGGTCGTCTGTTTACGTCTGAGTC 38
2 ATAGCGTCGCAAATTTTGTCAGGATGGCCTTCCGTAACAGACTCAGACGTAAACAGACGA 60
3 TGACAAAATTTGCGACGCTATCTCTGACTCTGTACTGGATGCACTCCTGGCGGCAGACCC 60
4 ACCTGACCGGTGGTTACGAGAGTCTCAACCGCAACACGAGAACGAGGGTCTGCCGCCAGG 60
5 CGTAACCACCGGTCAGGTTCACGTTGTTGGTGAAGTTACCACCTCTGCAAAAGAAGCGTT 60
6 TTCCAGAATACGCGCACGGACCGTGTTAGTGATATCCGCAAACGCTTCTTTTGCAGAGGT 60
7 CGTGCGCGTATTCTGGAAATTGGCTACGACTCTTCTGACAAGGGTTTCGATGGTGCGACG 60
8 AATATCAGGAGACTGCGCACCGATACCGATGTTCACGCCGCACGTCGCACCATCGAAACC 60
9 GTGCGCAGTCTCCTGATATTGCGCAGGGTGTTGACACCGCACACGAAGCGCGTGTGGAAG 60
10 CCCTGGTCACCGGCACCCTGAGAGTCCAGAGGATCTGCAGCACCTTCCACACGCGCTTCG 60
11 GTGCCGGTGACCAGGGCCTGATGTTCGGTTACGCAATCAATGCGACCCCTGAACTGATGC 60
12 TGAGACGACGAGACAGGCGATGGGCCAGCGCGATCGGGAGCGGCATCAGTTCAGGGGTCG 60
13 GCCTGTCTCGTCGTCTCACTGAGGTTCGTAAAAACGGTGTTCTCCCGTACCTCCGCCCAG 60
14 CGGAACGTTATCCTCGTACGCGATAGTAACCTGGGTCTTACCATCTGGGCGGAGGTACGG 60
15 CGTACGAGGATAACGTTCCGGTTCGTCTCGACACTGTGGTAATCTCTACCCAGCACGCGG 60
16 CTCGCGGATGTCCGGATCGAGGGTTTTCTCCAGGTCGATGTCAGCCGCGTGCTGGGTAGA 60
17 ATCCGGACATCCGCGAGAAAGTTCTCAACACTGTCCTGGATGACCTCGCACATGAGACGC 60
18 TTGCCCGTAGGATTTACCAGAACACGAACGGTGGATGCGTCCAGCGTCTCATGTGCGAGG 60
19 CTGGTAAATCCTACGGGCAAATTCGTTCTGGGTGGTCCGATGGGTGATGCGGGTCTGACT 60
20 ACGGGCCCAACCGCCGTAGGTGTCTACGATGATCTTGCGACCAGTCAGACCCGCATCACC 60
21 GCGGTTGGGCCCGTCACGGTGGTGGTGCGTTCAGCGGCAAAGACCCGTCTAAGGTTGACC 60
22 GACGTTTTTCGCAACCCAACGCATGGCATAGGCCGCAGAACGGTCAACCTTAGACGGGTC 60
23 GTTGGGTTGCGAAAAACGTCGTGGCAGCAGGTCTGGCGGAGCGTGTAGAAGTTCAAGTCG 60
24 CGAACAGACCAACAGGTGCCGCTTTACCGATTGCATACGCGACTTGAACTTCTACACGCT 60
25 GCACCTGTTGGTCTGTTCGTTGAAACTTTCGGCACCGAAACGGAAGATCCGGTAAAAATC 60
26 CCGGACGCAGATCAAAAACCTCACCAATCGCTTTTTCGATTTTTACCGGATCTTCCGTTT 60
27 GTTTTTGATCTGCGTCCGGGTGCCATCATTCGTGATCTGAATCTGCTCCGTCCAATCTAC 60
28 GTCGGTACGACCAAAGTGGCCGTACGCTGCGGTCGGCGCGTAGATTGGACGGAGCAGATT 60
29 CCACTTTGGTCGTACCGACGTTGAACTGCCGTGGGAACAACTGGACAAGGTCGACGATCT 60
30 TTAGATCGCACGTTTGAGATCGTCGACCTTGTCCA 35
*GC% Content for gene (codon optimized):
55.7% *had
1753 bases
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
From Tuberculist (NCBI)
VSEKGRLFTSESVTEGHPDKICDAISDSVLDALLAADPRSRVAVETLVTTGQVHVVGEVT
TSAKEAFADITNTVRARILEIGYDSSDKGFDGATCGVNIGIGAQSPDIAQGVDTAHEARV
EGAADPLDSQGAGDQGLMFGYAINATPELMPLPIALAHRLSRRLTEVRKNGVLPYLRPDG
KTQVTIAYEDNVPVRLDTVVISTQHAADIDLEKTLDPDIREKVLNTVLDDLAHETLDAST
VRVLVNPTGKFVLGGPMGDAGLTGRKIIVDTYGGWARHGGGAFSGKDPSKVDRSAAYAMRWVAKNVVAAGLAERVEVQVAYAIGKAAPVGLFVETFGTETEDPVKIEKAIGEVFDLRPGA
IIRDLNLLRPIYAPTAAYGHFGRTDVELPWEQLDKVDDLKRAI
Methionine added to beginning of sequence for E coli
MVSEKGRLFTSESVTEGHPDKICDAISDSVLDALLAADPRSRVAVETLVTTGQVHVVGEVT
TSAKEAFADITNTVRARILEIGYDSSDKGFDGATCGVNIGIGAQSPDIAQGVDTAHEARV
EGAADPLDSQGAGDQGLMFGYAINATPELMPLPIALAHRLSRRLTEVRKNGVLPYLRPDG
KTQVTIAYEDNVPVRLDTVVISTQHAADIDLEKTLDPDIREKVLNTVLDDLAHETLDAST
VRVLVNPTGKFVLGGPMGDAGLTGRKIIVDTYGGWARHGGGAFSGKDPSKVDRSAAYAMR
WVAKNVVAAGLAERVEVQVAYAIGKAAPVGLFVETFGTETEDPVKIEKAIGEVFDLRPGA
IIRDLNLLRPIYAPTAAYGHFGRTDVELPWEQLDKVDDLKRAI
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
5’ TACTTCCAATCCATGGTTTCTGAAAAAGGTCG 3’ 32 bp
GC Content 40.6%
0 mM Mg2+ Tm 60.3oC1.5 mM Mg2+ Tm 68.1oC 2 mM Mg2+ Tm 68.5oC
4 mM Mg2+ Tm 69.4oC 6 mM Mg2+ Tm 69.9oC
Reverse Primer:
5’ TATCCACCTTTACTGTTAGATCGCACGTTTGAGAT 3’ 32 bp
GC Content 40%
0 mM Mg2+ Tm 60.7oC1.5 mM Mg2+ Tm 68.6oC 2 mM Mg2+ Tm 69oC
4 mM Mg2+ Tm 69.8oC 6 mM Mg2+ Tm 70.3oC
References:
[1] Ducati, R. G.; Ruffino-Netto, A.; Basso, L. A.; Santos, D. S., The resumption of consumption -- a review on tuberculosis. Mem Inst Oswaldo Cruz 2006,101 (7), 697-714.